Device for measuring substrate concentration

ABSTRACT

The present invention provides a method for measuring a substrate concentration by accumulating an energy resulting from a reaction between a biocatalyst and a substrate recognized by the biocatalyst to a certain level; and using a dependency of an accumulation rate on the substrate concentration as an index; and a apparatus therefor. In particular, the present invention provides a method in which the measurement of the accumulation rate is carried out by measuring a frequency of an energy release in a certain amount of time when the energy accumulated in the capacitor reaches the certain level and is then released.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/678,475, filed Mar. 16, 2010 which is incorporated herein byreference and which is the U.S. National Phase under 35 U.S.C. §371 ofInternational Application PCT/JP2008/002575, filed Sep. 18, 2008, whichwas published in a non-English language, which claims priority to JP2007-241333, filed Sep. 18, 2007.

TECHNICAL FIELD

The present invention relates to a method for measuring theconcentration of a substrate and a device (biosensor) therefor.

BACKGROUND ART

A biosensor is a sensor for measuring a substrate, by a biocatalyticreaction, namely by allowing a biocatalyst to react with a compound, asubstrate thereof, which sensor has, as a transducer, a device capableof detecting a product resulting from the biocatalytic reaction, adecreased substrate or a chemical compound generated by a reaction withthe product. Or, it means a sensor for measuring the substrate havingalso, as a transducer, a device capable of detecting physical signalssuch as a change in light and/or color, or fluorescence, resulting fromthe biocatalytic reaction. Examples of the biocatalyst include enzymes,organelles, cells and microorganisms.

That is, it can be said that a biosensor is a sensor which converts abiocatalytic reaction into a signal which an electronic device candetect, by using a biocatalyst as a molecular recognition element and bycombining a signal thereof with a transducer such as an electrochemicaldevice, optical device or heat detection device, and thereby is capableof analyzing a substrate recognized by the biocatalyst. One of therepresentative biosensors is an enzyme sensor using an enzyme as abiocatalyst. For instance, for the purpose of measuring glucose (grapesugar), a glucose sensor has been developed, on the basis of a concept,wherein an enzyme oxidizing glucose is immobilized on the surface of anelectrode such as an oxygen electrode and a hydrogen peroxide electrode,and the amount of oxygen consumed by an oxidative reaction of glucoseand the amount of hydrogen peroxide generated at the same time areelectrochemically measured.

Among enzyme sensors widely used at present, a sensor using anoxidoreductase is mainly used. A major principle thereof is based on amethod for measuring, with an ampere meter, electrons generated when areduced substance generated by an enzyme reaction at an anode isre-oxidized by an electric potential externally applied; or a method formeasuring by a difference in the electric potentials generated betweenan anode and a cathode when the generated electrons are reduced at thecathode.

Also, as a method used in a simple blood sugar diagnostic apparatus orthe like, a method comprising colorizing a reduced substance generatedby an enzyme reaction such as hydrogen peroxide or a reduced artificialelectron acceptor in accordance with a conventional method, anddetermining the color by an optical sensor has been employed.

In addition, as an example of a special enzyme, an enzyme sensoremploying a luciferase as an enzyme, which is an enzyme derived from alight-emitting organism such as a firefly, has been also reported, whichenzyme sensor is characterized by detecting light generated by an enzymereaction where a substrate for the luciferase reacts. Yet, as for thismethod, applications are limited to the cases where a luciferase can beused, such as the cases where the objective is limited to the detectionof a substance which is an substrate for the luciferase, such as ATP, orthe cases where there can be employed a principle that when an antibodyreaction is detected, it can be indirectly detected via an opticalsignal by labeling the antibody with the luciferase.

Non-patent Literature 1: Katz et al., J. Am. Chem. Soc. 2001, 123,10752-10753

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

In the conventional biosensor described above, a measuring apparatususing a biosensor such as an enzyme sensor (hereinafter referred to asenzyme sensor system) is composed of a measuring part by the biosensorand a monitor part in which a measured signal is received and processed.In major types, these parts are integrated or, as seen in aself-measuring blood sugar diagnostic apparatus, a part corresponding tothe measuring part can be detached to be disposable. Also, the measuringpart for detecting a substrate concentration and the monitor part fordetecting a signal from the biosensor need to be in direct contact withor be wired to a field where the biocatalytic reaction takes place, orrequire a circuit for actuating a special transmitter and a power supplytherefore to be provided, all of which are problematic.

Meanwhile, thus far, an enzyme sensor using an electromotive force of anenzyme fuel cell as an index has been reported. Yet, since theelectromotive force of this fuel cell alone is less than 1 V, by theelectromotive force of the fuel cell, the fuel cell was not able tooperate, as is, a device for sensing. In the case of measuring asubstrate concentration using the electromotive force of the fuelcell-type enzyme sensor as an index, it was required that theelectromotive force be directly connected to a voltmeter to measure theelectric potential, thereby measuring the substrate concentration. Or,it was required that the electromotive force be directly connected tothe voltmeter for measuring the electromotive force, and a responsevalue of the voltmeter be transmitted to an external receiver by awireless device actuated by an external power supply. (See “A NovelWireless Glucose Sensor Employing Direct Electron Transfer PrincipleBased Enzyme Fuel Cell”, Noriko Kakehi, Tomohiko Yamazaki, WakakoTsugawa and Koji Sode Biosensors & Bioelectronics Epub 2006 Dec. 11).

Thus, although a fuel cell-type enzyme sensor is compact and has a highperformance sensing ability, in cases where it is embedded in or mountedon a living body and its signal is attempted to be detected in wireless,data of the fuel cell-type enzyme sensor cannot be read without furtherproviding a power supply. Therefore, in order to increase theelectromotive force of an enzyme fuel cell, it is considered that, byconnecting the enzyme fuel cells in series, the electromotive force canbe increased depending on the number of the fuel cells. However, incases where power generation in a living body or monitoring in a livingbody is aimed, disposing the enzyme fuel cells connected in series inthe living body complicates the apparatus and requires large electrodes,which were problematic and practically impossible.

Means for Solving the Problems

Therefore, in the present invention, it is proposed that the measurementbe carried out by accumulating energy resulting from a biocatalyticreaction such as an enzyme and using, as an index, the accumulation rateor the frequency with which the energy once accumulated is released.Thus, in the present invention, in cases where a certain amount ofenergy is produced by a biocatalytic reaction depending on the substrateconcentration, by paying attention that the production rate thereofdepends on the substrate concentration, it is used the fact that if theenergy to be accumulated is set to a certain level and the energy isreleased when the energy is accumulated to the certain level, thefrequency of the release depends on the substrate concentration of thebiocatalyst. The present invention proposes that the substrateconcentration of the biocatalyst be measured by measuring the frequencyof the release.

In particular, it is able to provide a method for measuring theconcentration of a substrate and a device therefor by combining abiocatalyst such as an enzyme and a device having a circuit in which, byaccumulating an electrical energy in a capacitor up to a certain levelas energy resulting from a biocatalytic reaction and releasing theenergy, a signal is generated depending on the amount of the releasedelectricity.

Also, by combining a circuit which generates light, sonic waves,electromagnetic waves or the like from the electrical energy accumulatedin the capacitor, a signal generated from the sensor is able to bereadily received by a signal detector in a non-contact monitor part.Therefore, the measuring part and monitor part can be separated. Thus,the separation of the measuring part and monitor part allows themeasuring part to be much smaller. Such miniaturization is advantageousin a portable sensor, or a sensor placed or embedded inside a body.

Furthermore, in another aspect of the present invention, provided is acircuit of a novel wireless enzyme sensor capable of accumulating anelectrical energy generated by an enzyme reaction in a capacitor,actuating a wireless device by the electromotive force, and transmittingthe signal to an external receiver. Thus, provided is a self-propelledwireless enzyme sensor capable of transmitting a signal of a sensor bywireless using an electromotive force of an enzyme fuel cell without apower supply. Also, in the device of the present invention, unlike aconventional measurement of an electric potential or the like, awireless signal can be detected by a receiving side when theelectromotive force exceeds the actuation voltage of a wirelesstransmitter. Based on this, a device for measuring the concentration ofa substrate using the actuation frequency of the wireless transmitter asan index is provided.

The structure of the present invention is as follows:

-   -   (1) A method for measuring the concentration of a substrate, the        method containing:        -   accumulating energy resulting from a reaction between a            biocatalyst and a substrate recognized by the biocatalyst to            a certain level; and        -   measuring the substrate concentration by using as an index            the fact that the accumulation rate of the energy is            dependent on the substrate concentration.    -   (2) The method according to above item 1, wherein said index is        measured on the basis of the release frequency of the energy in        a given period wherein said energy is released when reaching or        exceeding said certain level.    -   (3) The method according to above item 1 or 2, wherein the        biocatalyst is an enzyme, an organelle, a microorganism or a        cell.    -   (4) The method according to any one of above items 1 to 3,        wherein the reaction catalyzed by the biocatalyst is an        oxidation reaction.    -   (5) The method according to above item 3, wherein the        biocatalyst is an enzyme.    -   (6) The method according to above item 5, wherein the enzyme is        an oxidoreductase.    -   (7) The method according to any one of above items 1 to 6,        wherein the energy to be accumulated is accumulated in a        capacitor as an electrical charge.    -   (8) An apparatus for measuring the concentration of a substrate,        the apparatus containing:        -   a fuel cell having an anode on which a substrate of a            biocatalyst is disposed and a cathode on which an external            electron acceptor is disposed;        -   a capacitor connected to the fuel cell in series; and        -   a measuring device for measuring the substrate concentration            by using the charging rate of the capacitor as an index;        -   wherein an electromotive force generated by transferring            electrons generated by a reaction between the substrate and            the biocatalyst into the external electron acceptor on the            cathode is charged in the capacitor, and the charging rate            thereof is measured by the measuring device.    -   (9) The apparatus according to above item 8, wherein the        measuring device measures the discharging frequency, by said        capacitor discharging an accumulated electric potential when the        electric potential charged in the capacitor reaches or exceeds a        certain level.    -   (10) The apparatus according to above item 8 or 9, further        containing a charge pump for charging the capacitor, which        charge pump boosts the electromotive force based on the        biocatalytic reaction when the capacitor is charged.    -   (11) The apparatus according to above item 9 or 10, wherein the        measuring device has a signal generation circuit generating a        signal by the discharging from the capacitor and measures the        frequency of the signal.    -   (12) The apparatus according to above item 11, wherein the        signal generation circuit is a wireless transmitter.    -   (13) The apparatus according to above item 11 or 12, wherein the        measuring device measures a physical signal and/or a chemical        signal generated when the signal generation circuit is actuated.    -   (14) The apparatus according to above item 13, wherein the        physical signal and/or the chemical signal are/is a sonic wave,        light or an electromagnetic wave.    -   (15) The apparatus according to any one of above items 11 to 14,        wherein the measuring device further contains a receiver for        receiving the signal generated by the discharging of the        capacitor when the capacitor exceeds the actuation voltage of        the wireless transmitter by charging.    -   (16) The apparatus according to any of above items 8 to 15,        wherein the biocatalyst disposed on the anode is an enzyme.    -   (17) The apparatus according to above item 16, wherein the        enzyme is an oxidoreductase.    -   (18) The apparatus according to above item 16, wherein the        enzyme catalyzes oxidation of glucose.

As the biocatalyst used in the present invention, enzymes, organelles,cells, microorganisms and the like can be used. Also, as a reactioncatalyzed by the biocatalyst, a redox reaction of an object to bemeasured is preferred. As the enzyme, various oxidoreductases can beused. Examples thereof include oxidases for alcohol, glucose,cholesterol, fructosyl amine, glycerin and uric acid, which oxidases useFAD as a coenzyme; dehydrogenases for alcohol, glucose and glycerin,which dehydrogenases require FAD as a coenzyme; and dehydrogenases foralcohol, glucose and glycerin, which dehydrogenases use PQQ as acoenzyme. In particular, in cases where glucose is to be measured, aglucose oxidase and/or glucose dehydrogenase using FAD or PQQ as acoenzyme is/are preferred. This may be an enzyme isolated and purifiedfrom a microorganism or cells which produce the enzyme. Or, it may be arecombinant enzyme produced in E. coli or the like.

In addition, the biocatalyst used in the present invention may not be anenzyme alone, but may be a membrane containing the enzyme, an organellecontaining the enzyme or a cell containing the enzyme as long as it isable to oxidize a substrate at an anode and transmit this electron to anappropriate electron acceptor or directly to an electrode, and can beused as long as an oxidation reaction of the above-mentioned substrateis achieved by the result of these enzyme reactions and a plurality ofenzyme reactions coupled with them.

As the method for generating an electrical energy using an enzymeaccording to the present invention, an enzyme fuel cell can be employed.That is, it is an enzyme fuel cell characterized in that an oxidase ordehydrogenase is immobilized on an anode.

In this case, as a cathode, an electrode in which an enzyme reducingoxygen such as bilirubin oxidase is used or an electrode in whichappropriate electron acceptors are combined can be used. Or, a catalysthaving ability to reduce oxygen such as platinum or an inorganiccatalyst containing platinum can be used.

Also, a structure containing an electron acceptor as well as the enzymeis thought to be used as an anode. That is, those transferring anelectron obtained by an enzyme reaction to an artificial electronacceptor and oxidizing the electron on the electrode can be used. Or,dehydrogenases capable of directly transferring electrons to anelectrode such as an enzyme having cytochrome in an electron transportsubunit and the like can constitute the anode without adding anyartificial electron acceptor. As electrode materials for the anode andcathode, electrodes filled or coated with carbon particles, carbonelectrodes, gold electrodes, platinum electrodes, or the like can beused.

The artificial electron acceptor of the anode or cathode is notparticularly restricted and an osmium complex, a ruthenium complex,phenazine methosulfate and a derivative thereof, a quinone compound orthe like can be used.

The enzyme for the cathode is not particularly restricted, and bilirubinoxidase or laccase can be applied. The artificial electron acceptor ofthe cathode is not restricted and potassium ferricyanide, ABTS or thelike can be used.

As the enzyme for the anode, various oxidases or dehydrogenase can beused. In particular, in cases where glucose is to be measured, glucoseoxidase or glucose dehydrogenase using PQQ or FAD as a coenzyme can beused.

In the present invention, as a method for mounting an enzyme on anelectrode, a mixture of the enzyme as is and an electrode material suchas carbon paste can be used. Or, after preparation by a general methodfor immobilizing an enzyme, an immobilized enzyme can be mounted on theelectrode. Examples include methods in which a cross-linking treatmentwith a binary cross-linking reagent such as glutaraldehyde is carriedout after the mixing of both; and methods of inclusively immobilizing ina synthetic polymer such as a photocrosslinking polymer,electroconductive polymer or redox polymer, or a natural polymer matrix.The thus prepared mixed protein is mixed with carbon particles or mixedwith carbon paste which is composed of the carbon particles and is in amode of being readily combined with an enzyme. Thereafter, the resultingmixture is further subjected to a cross-linking treatment and thenmounted on the electrode composed of carbon, gold or platinum. As thecarbon particle, one having a specific surface area ranging from about10 m²/g to not less than 500 m²/g, more preferably not less than 800m²/g can be used. Examples of the former include VULCAN™ (CabotCorporation) as a commercially available product and examples of thelater include Ketchen black™ (Akzo Nobel Chemicals Inc.).

Further, when the enzyme is mounted on the electrode in this manner, anartificial electron acceptor can be immobilized at the same time.Typically, glucose dehydrogenase using FAD as a coenzyme, FADGDH andmethoxy phenazine methosulfate (mPMS) are mixed. The mixture is furthermixed with carbon paste and then freeze dried. This is mounted on acarbon electrode and the resultant is immersed as is in a glutaraldehydeaqueous solution to cross-link a protein, thereby making an enzymeelectrode.

In the enzyme fuel cell, an oxidase or dehydrogenase using a measuringobject as a substrate is immobilized on the anode electrode. An oxygenreductase is immobilized on the cathode. The thus prepared electrodesare used as electrodes for the anode and the cathode. In the anode, forexample, m-PMS can be used as an artificial electron acceptor and alsoin the cathode, for example, ABTS can be used as an artificial electronacceptor.

By connecting the thus prepared part where an electrical energy isgenerated by an enzyme reaction to a capacitor, an electrical energy canbe stored. That is, based on an electromotive force obtained by theenzyme reaction, the capacitor connected to the circuit shown in FIG. 2is charged until the capacity of the capacitor is filled up. Therefore,in cases where the enzyme reaction is carried out in a solution of thesame substrate concentration, when a capacitor with a larger capacity isused, it takes a longer period of time to complete charging. Conversely,when a capacitor with a smaller capacity is used, the time to completecharging is shorter. Or, in cases where a capacitor with the samecapacity is used, when the substrate concentration of the enzyme islower, the amount of the electrical energy generated per unit time issmaller and thus the time to complete charging is longer, whereas whenthe substrate concentration is higher, the time to complete charging is,in contrast, shorter. That is, by setting the capacitor at a certainamount of capacity, the time required for charging varies depending onthe concentration of the substrate of the enzyme. Therefore, thesubstrate concentration can be measured by using the time required forcharging (charging rate) as an index. That is, by recording in advancethe correlation between the observed time required for charging(charging rate) and the substrate concentration and, based on that,preparing a calibration curve, the substrate concentration of an unknownsample can be measured from the observed time required for charging.

Also, by connecting an appropriate circuit to the capacitor such thatdischarging begins when charging is completed, the substrateconcentration can be measured in the same manner by measuring thecharging and discharging frequency per unit time. That is, by recordingin advance the correlation between the observed discharging frequencyper unit time and the substrate concentration and preparing, based onthat, a calibration curve, the substrate concentration of an unknownsample can be measured from the observed discharging frequency.

Further, if light, a sonic wave or an electromagnetic wave is generatedby a circuit connecting there, by observing the light, the sonic wave orthe electromagnetic wave, and measuring the frequency at which it isobserved per unit time or the interval between its observations, theconcentration of the substrate can be measured. That is, by recording inadvance the correlation between the observed time required forgenerating the light, the sonic wave or the electromagnetic wave and thesubstrate concentration and preparing, based on that, a calibrationcurve, the substrate concentration of an unknown sample can be measuredfrom the observed time required for generating the light, the sonic waveor the electromagnetic wave or an frequency per unit time.

In addition, depending on a circuit to be actuated, it is possible thatan electric potential of the capacitor is appropriately set. That is, bycombining an electromotive force of an enzyme fuel cell which generatesan electrical energy generated by an enzyme reaction as an electromotiveforce with a booster circuit, the electric potential charged in thecapacitor can be increased. For this boosting, a commercially availablecharge pump or an IC circuit thereof can be used. The electric potentialstored in the capacitor can be adjusted depending on the type and thenumber of the charge pump combined. The electric potential in thecapacitor can be set depending on the signal generation circuit to beactuated.

The frequency at which the capacitor is charged and discharged depends,as described above, on the capacity of the capacitor and theconcentration of the substrate. That is, if the substrate concentrationis constant, the smaller the capacity of the capacitor is, the higherthe charging and discharging frequency is. And, the larger the capacityof the capacitor is, the lower the charging and discharging frequencyis. Also, if the capacity of the capacitor is constant, the charging anddischarging frequency changes depending on the substrate concentration.And, the lower the substrate concentration is, the lower the chargingand discharging frequency is. The higher the substrate concentration is,the higher the charging and discharging frequency is.

For instance, when a voltmeter is connected to both ends of thecapacitor, what is observed is shown in FIG. 3. In this mode, a sampleof a constant concentration of glucose is used as a substrate; an enzymecatalyzing dehydrogenation of glucose is employed as an enzyme; and anelectromotive force generated by an enzyme fuel cell is boosted from 0.3V of the enzyme fuel cell to 1.8 V through a charge pump, therebycharging the capacitor. As shown in FIG. 3, it can be observed that theelectrical potential of the capacitor reaches 1.8 V at regularintervals, and the electrical energy generated from the enzyme reactionis stored and then released. In this case, when the capacity of theconnected capacitor is changed from 0.47 g to 1 μF, the observedcharging and discharging interval changes. That is, when the capacity ofthe capacitor is 0.47 μF, the interval was 0.2 seconds (the charging anddischarging frequency 5 times/second, 5 Hz) whereas the frequencychanges as follows: 2.4 Hz at 1 μF, 0.27 Hz at 10 μF and 0.028 Hz at 100μF.

Further, when a condition where the capacitor is charged, by using acapacitor of 10 μF and changing the concentration of glucose, isobserved, the charging and discharging interval is longer at a lowerconcentration of glucose, and the charging and discharging interval isshorter at a higher concentration of glucose (see FIG. 4). Conversely,when this is observed as a charging and discharging frequency, thecharging and discharging frequency is lower at a lower concentration ofglucose, and the charging and discharging frequency is higher at ahigher concentration of glucose.

By connecting this circuit to a circuit generating a signal depending oncharging and discharging of the capacitor in the same manner andobserving the light, the sonic wave or the electromagnetic wavegenerated from there, the substrate concentration can be measured in thesame manner. For example, in cases where a light emitting diode isconnected, by observing the emission interval of the light emittingdiode or the emission frequency, the substrate concentration can bemeasured.

As shown in FIG. 5, the emission interval is longer at a lowerconcentration of glucose and the emission interval is shorter at ahigher concentration of glucose. Conversely, when this is observed as anemission frequency, the emission frequency is lower at a lowerconcentration of glucose and the emission frequency is higher at ahigher concentration of glucose.

Also, in cases where a resonant circuit generating an electromagneticwave is connected to this circuit, by observing the interval or thefrequency of the transmission of the electromagnetic wave, the substrateconcentration can be measured. In this case, the interval of thetransmitted electromagnetic wave is longer at a lower concentration ofglucose and the interval is shorter at a higher concentration ofglucose. When this is observed as a transmission frequency of theelectromagnetic wave, the transmission is lower at a lower concentrationof glucose and the transmission frequency is higher at a higherconcentration of glucose.

As can be seen from such a mode, it is evident that, when a signaltransmitter actuated by the capacitance and electrical potential of acapacitor is connected, regardless of the type of the signal transmittedfrom there, that is, light, a sonic wave, or an electromagnetic wave,the concentration, of a substrate of an enzyme reaction can be measuredby observing the interval and frequency. In addition, it is also evidentthat the enzyme is not limited to the dehydrogenase using glucose as asubstrate which is shown herein, and various oxidases and dehydrogenasescan be used. Examples thereof include oxidases for alcohol, glucose,cholesterol, fructosyl amine, glycerin and uric acid, which oxidases useFAD as a coenzyme; dehydrogenases for alcohol, glucose and glycerin,which dehydrogenases use FAD as a coenzyme; and dehydrogenases foralcohol, glucose and glycerin, which dehydrogenases use PQQ as acoenzyme. Even when it is not an enzyme alone, as long as it is able tooxidize a substrate at an anode and transmit this electron to anappropriate electron acceptor or directly to an electrode, it may be amembrane, an organelle, a cell or a microorganism, all of which containthe enzyme. If an oxidation reaction of the above-mentioned substrate isachieved by the result of these enzyme reactions, it can be used, whichis self-explanatory from study cases of biosensors using biocatalystscatalyzing various redox reactions.

Also, as another mode, a transmission circuit used in wirelesscommunication can be used as a signal transmission circuit connected toa capacitor. These transmission circuits require a certain level orhigher of electric potential for its actuation. If the electromotiveforce is below this level, the circuit stops and so does thetransmission. Also, it cannot be actuated when the electromotive forceis not higher than the certain level. That is, when the wirelesstransmission circuit actuated at 1.5 V is connected to the capacitor andthe transmitted signal is observed by a distant reception system, thetransmission from the wireless is observed corresponding to charging anddischarging of the capacitor. That is, depending on the concentration ofa substrate of an enzyme reaction, the wireless transmission circuit isactuated and the signal is transmitted. The interval is longer when theenzyme substrate concentration is lower and the interval is shorter whenthe enzyme substrate concentration is higher. Also, the transmissionfrequency of the signal is lower when the enzyme substrate concentrationis lower and the transmission frequency of the signal is higher when theenzyme substrate concentration is higher. Therefore, the substrateconcentration of enzyme can be measured by observing a receivedtransmission record.

As such a wireless transmission circuit, a resonant circuit may beemployed. Also, for a capacitor in this resonant circuit, a capacitorwhose capacity is variable may also be employed.

BRIEF DESCRIPTION OF THE DRAWINGS

[FIG. 1] FIG. 1 shows a schematic view of the method for measuring asubstrate and the apparatus therefor according to the present invention.

[FIG. 2] FIG. 2 shows a circuit diagram of a capacitor used in thepresent invention.

[FIG. 3] FIG. 3 shows the change in the electric potential by repeatedcharging and discharging in a capacitor in the apparatus according tothe present invention.

[FIG. 4] FIG. 4 shows the relationship between the charging frequency ofa capacitor and the concentration of glucose.

[FIG. 5] FIG. 5 shows the emission interval (time) of a light emittingdiode for the change in the concentration of glucose.

[FIG. 6A] FIG. 6A shows the time required for charging of a capacitor(resistance 100 kΩ).

[FIG. 6B] FIG. 6B shows the time required for charging of a capacitor(resistance 500 kΩ).

[FIG. 6C] FIG. 6C shows the time required for charging of a capacitor(resistance 500 kΩ).

[FIG. 7A] FIG. 7A shows the time required for charging of a capacitorfor the change in the concentration of glucose (10 kΩ).

[FIG. 7B] FIG. 7B shows the time required for charging of a capacitorfor the change in the concentration of glucose (500 kΩ).

[FIG. 8A] FIG. 8A shows the change in the electric potential of acapacitor by an enzyme fuel cell for time (0.47 μF).

[FIG. 8B] FIG. 8B shows the change in the electric potential of acapacitor by an enzyme fuel cell for time (10 μF).

[FIG. 8C] FIG. 8C shows the change in the electric potential of acapacitor by an enzyme fuel cell for time (10 μF).

[FIG. 8D] FIG. 8D shows the change in the electric potential of acapacitor by an enzyme fuel cell for time (100 μF).

[FIG. 9] FIG. 9 shows the signal frequency in the case of changing theconcentration of glucose.

[FIG. 10] FIG. 10 shows the change in the time required for a capacitorreaching 1.8 V for the change in the concentration of glucose.

[FIG. 11] The correlation of the frequency at which a capacitor reaches1.8 V per unit time for the change in the concentration of glucose.

[FIG. 12] The correlation between the frequency of an observed signaland the concentration of glucose in a wireless sensor (1.8 V boosting).

[FIG. 13] The correlation between the frequency of an observed signaland the concentration of glucose in a wireless sensor (2.4 V boosting).

[FIG. 14] FIG. 14 shows an example, as a signal transmission circuit, ofa measurement/transmission circuit using a resonant circuit as atransmitter.

[FIG. 15] FIG. 15 shows an example in which an electromagnetic waveobserved by using the transmission circuit shown in FIG. 14 wasrecorded.

[FIG. 16] FIG. 16 shows an example, as a signal transmission circuit, ofa measurement/transmission circuit using, as a transmitter, a resonantcircuit using a varicap diode.

[FIG. 17] FIG. 17 shows an example in which an electromagnetic waveobserved by using a transmission circuit shown in FIG. 16 and theconcentration of glucose in a sample were measured.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will now be described in detail below by way ofexamples thereof. However, the present invention is not limited to theexamples.

Example 1 Preparation of Anode:

Ketchen black ink (10 mL), 100 mM PPB (pH 7.0) (10 mL) and an FADGDHcomplex solution (40 mL) (1.2 U/mL) were mixed. And, 50 mL of themixture was coated uniformly on 1 cm² of carbon cloth and then air-driedat 4° C. for 3 hours. By immersing the resultant in a 1% glutaraldehydesolution (10 ml) at room temperature for 30 minutes, a cross-linkingtreatment was carried out. Next, by immersing this carbon cloth in 10 mMTris-HCl (10 ml) for 20 minutes, unreacted glutaraldehyde was removed.Thereafter, the resultant was immersed in 100 mM PPB (pH 7.0) (10 ml)for 1 hour to be equilibrated, thereby preparing an anode.

Preparation of Cathode:

A platinum supported carbon ink (10 mL) and 100 mM PPB (pH 7.0) (50 mL)were mixed. And, 50 mL of the mixture was coated uniformly on carboncloth and the resultant was air-dried at 4° C. for 3 hours. To theresultant, 50 mL of 3% (w/v) Poly(dimethylsiloxane) (PDMS) diluted byethanol was dropped and air-dried overnight to provide a cathode.

Construction of Electrical Cell and Circuit:

Using the prepared anode and cathode, an electrical cell was constructedusing 100 mM PPB (pH 7.0) containing 20 mM glucose as a reactionsolution. The electrical cell, a variable resister, a capacitor and aswitch were all connected in series, thereby preparing a circuit.

In the thus prepared circuit, the charging time of the capacitor wasexamined. Thus, using two types of capacitors (0.1 mF and 1 mF), underthe condition where charging was carried out at a glucose concentrationof 20 mM, an electric potential applied to the capacitor and an electriccurrent which flows in the circuit when the switch were connected to aresistance were measured to examine the charging time for the capacitor.

In this case, the results when the capacitor of 1 mF was used as thecapacitor and the resistance was 100 kΩ, 500 kΩ and 1000 kΩ were shownin FIG. 6. The electric current flowed at the same time when the switchwas turned on. Also, the electric current decreased over time and theelectric potential on the capacitor increased. Further, when theresistance value was changed to decrease or increase the electriccurrent value, the charging time of the capacitor increased or decreasedaccordingly. At any resistance, when the charge amount charged in thecapacitor was calculated from the electric current value which flowed inthe circuit, it was almost equal to the capacity of the capacitor. Inaddition, when the capacitor of 0.1 mF was used, the similar resultswere obtained. Yet, as compared with the capacitor of 1 mF, the chargingtime was shorter. It was shown that, even in cases where an enzyme fuelcell was used as a power supply, the capacitor was able to functionadequately.

Using a circuit prepared in the same way, a dependency of the chargingtime of a capacitor on the glucose concentration was evaluated. Using acondenser of 1 mF, the electric potential on the capacitor was measuredwhen a resistance was set to 10 kΩ or 500 kΩ. In this case, by graduallyadding a glucose sample to increase the concentration of glucose in areaction solution, the charging time at each concentration of glucosewas examined.

The results are shown in FIG. 7. At a resistance of either 10 kΩ or 500kΩ, as the glucose concentration increases, the charging time decreased.At 500 kΩ, the decrease in the charging time was scarcely observed withabout 6 mM whereas, at 10 kΩ, an increase in the electric current wasobserved up to 11 mM. It was shown that, using the time for charging thecapacitor as an index, the substrate concentration of an enzyme was ableto be measured.

Example 2 Preparation of Anode:

Ketchen black ink (10 mL), 100 mM PPB (pH 7.0) (10 mL) and an FADGDHcomplex solution (40 mL) (4.2 U/mL) were mixed. And, 300 mL of themixture was coated uniformly on 6 cm² of carbon cloth and then air-driedat 4° C. for 3 hours. By immersing the resultant in a 1% glutaraldehydesolution (10 ml) at room temperature for 30 minutes, a cross-linkingtreatment was carried out. Next, by immersing this carbon cloth in 10 mMTris-HCl (10 ml) for 20 minutes, unreacted glutaraldehyde was removed.Thereafter, the resultant was immersed in 100 mM PPB (pH 7.0) (10 ml)for 1 hour to be equilibrated, thereby preparing an anode.

Preparation of Cathode:

A platinum supported carbon ink (60 mL) and 100 mM PPB (pH 7.0) (300 mL)were mixed. And, 300 mL of the mixture was uniformly coated on carboncloth (6 cm²) and the resultant was air-dried at 4° C. for 3 hours. Tothe resultant, 300 mL of 3% (w/v) Poly(dimethylsiloxane) (PDMS) dilutedby ethanol was dropped and air-dried overnight to provide a cathode.

Construction of Electrical Cell and Circuit:

Using the prepared anode and cathode, an electrical cell was constructedusing 100 mM PPB (pH 7.0) as a reaction solution. A charge pump (boostIC; S-882Z18 manufactured by Seiko Instruments Inc.) capable of boostingfrom 0.3 V to 1.8 V was combined with this fuel cell, therebyconstructing a circuit shown in FIG. 1. And, as a signal generationcircuit, an orange light emitting diode was connected and variouscapacitors of 0.47 to 100 mF were further connected. By measuring theelectric potential on the capacitor, and the emission interval andfrequency of the light emitting diode, the charging and dischargingcycle was evaluated.

Construction of Circuit Using Boost IC and Evaluation of SignalFrequency by Capacity of Capacitor

Actuation of the present circuit by an enzyme fuel cell was evaluated byblinking of the light emission diode or by the change with time of theelectric potential of the capacitor. And, a difference of the signalfrequency obtained when the capacitor was replaced with ones of 0.47 to100 mF was also evaluated. The concentration of glucose in a reactionsolution was 20 mM.

The change with time of the electric potential of the capacitor in thiscase is shown in FIG. 8. When the capacitor of 0.47 mF was used,spike-like signals were observed at a frequency of five times per second(5/s). Also, at the same cycle, the blinking of the diode was observed.When the capacity of the capacitor was changed, the frequency of thesignal changed. The cycle was 2.4/s, 0.27/s and 0.028/s when thecapacitor of 1 mF, 10 mF and 100 mF was used, respectively. It was shownthat the frequency of the signal was able to be increased by making thecapacity of the capacitor smaller.

Dependency of Capacitor Charge and Discharge Cycle on GlucoseConcentration

When the concentration of glucose which was an enzyme substrate waschanged, the signal frequency obtained at that time was evaluated byblinking of the light emission diode or the change with time of theelectric potential of the capacitor. As the capacitor, a capacitor of acapacity of 10 mF was used. The results are shown in FIG. 9. As theglucose concentration increases, the time required for the capacitorreaching a maximum electric potential became shorter, and an increase inthe frequency at which it reached a peak electric potential per unittime was observed. Based on this result, the glucose concentration andthe blinking of LED, that is, the time required for the capacitorreaching 1.8 V (FIG. 10) and the number of the blinking of LED per unittime, that is, the frequency at which the capacitor reached 1.8 V perunit time (FIG. 11) were determined. As shown in these curves, it wasdemonstrated that, from the obtained curve, the blinking of LED, thatis, the time required for the capacitor reaching 1.8 V and the number ofthe blinking of LED per unit time, that is, the frequency at which thecapacitor reached 1.8 V per unit time were dependent on the glucoseconcentration. From these, it was shown that the concentration ofglucose was able to be measured by using the signal frequency as anindex and a novel biosensor which used charging and discharging of thecapacitor was constructed.

Actuation of Wireless System by Boosted Enzyme Fuel Cell

Using the prepared anode and cathode, an electrical cell was constructedusing 100 mM PPB (pH 7.0) as a reaction solution. A charge pump (boostIC; S-882Z18 manufactured by Seiko Instruments Inc.) capable of boostingfrom 0.3 V to 1.8 V or a charge pump (boost IC; S-882Z24 manufactured bySeiko Instruments Inc.) capable of boosting from 0.3 V to 2.4 V wascombined with this fuel cell, thereby constructing a circuit shown inFIG. 1. As a signal generation circuit, a wireless system transmitter(infrared transmission) was connected. That is, a power supply part ofthe wireless transmitter was connected to a signal generation circuitshown in FIG. 1, thereby constructing a biosensor using, as an index,the actuation of the wireless transmission system by an electricpotential discharged when the capacitor was charged. The concentrationof glucose in the reaction solution was 0 to 25 mM.

As a result, in the presence of glucose, the wireless system transmitterwas actuated and a signal was transmitted to a receiver at regularintervals.

FIG. 12 shows the correlation between the frequency of the signalobserved at 1.8 V boosting and the glucose concentration. FIG. 13 showsthe correlation between the frequency of the signal observed at 2.4 Vboost and the glucose concentration. As shown here, at either boost, thereception frequency of the signal correlates with the glucoseconcentration and, by monitoring this frequency, the glucoseconcentration can be measured. The measurable concentration of glucoseis, in either case, from 0.5 mM to 20 mM, which covers a range enough tomeasure a blood sugar value in diabetes mellitus. Thus, it is shown thatit can be well applied to a blood sugar diagnostic apparatus including acontinuous blood sugar diagnostic apparatus.

From this, it was shown that the wireless system was able to be actuatedby using the electromotive force accumulated by the result of the enzymereaction, which electromotive force is charged in the capacitor.Therefore, it was shown that, in the present novel biosensor, thewireless transmitter was applicable as the signal transmission circuit.

Construction of Measurement/Transmission Circuit Using Resonant Circuitas Transmitter

A fuel cell was constructed in the same manner as described in Example 1and a capacitor of 10 μF and a boost IC from 0.3 to 1.8 V were combinedwith this, thereby constructing a biocapacitor. As a power supply, anoutput power of the biocapacitor was connected to both ends, therebyproducing a Hartley oscillation circuit. Using this transmitter, thereception frequency of an electromagnetic wave can be measured by areception circuit. As a result, in the presence of glucose, as describedin FIG. 15, it was observed that the electromagnetic wave was receivedat regular intervals. It is self explanatory, from the description thusfar, that this reception frequency of the electromagnetic wave dependson the glucose concentration.

Construction of Measurement/Transmission Circuit Using as TransmitterResonant Circuit Using Varicap Diode

A fuel cell was constructed in the same manner as described in Example 1and a capacitor of 0.47 μF and a boost IC from 0.3 to 1.8 V werecombined with this, thereby constructing a biocapacitor. As a powersupply, an output power of the biocapacitor was connected to both theends of a varicap (1sV149), thereby producing a Hartley oscillationcircuit. Using this transmitter, the reception frequency of anelectromagnetic wave can be measured by a reception circuit. FIG. 17shows an example in which the frequency of the electromagnetic waveobserved when using this transmission circuit and the concentration ofglucose in samples were measured. As shown here, the concentration ofglucose can be measured by using the present circuit.

1. A wireless system comprising: a capacitor in which energy resultingfrom a reaction between a biocatalyst and a substrate recognized by saidbiocatalyst is accumulated; a wireless transmitter which generates asignal on the basis of the discharging of said energy from saidcapacitor; and a receiver for receiving said generated signal.
 2. Thewireless system according to claim 1, wherein said energy is accumulatedto a certain level and the accumulation rate of said energy is dependenton the concentration of said substrate.
 3. The wireless system accordingto claim 1, wherein said discharging frequency, which is a frequency atwhich said energy is released when reaching or exceeding said certainlevel, is dependent on the concentration of said substrate.
 4. Thewireless system according to claim 1, wherein said biocatalyst is anenzyme, an organelle, a microorganism or a cell.
 5. The wireless systemaccording to claim 1, wherein said reaction catalyzed by saidbiocatalyst is an oxidation reaction.
 6. The wireless system accordingto claim 4, wherein said biocatalyst is an enzyme.
 7. The wirelesssystem according to claim 6, wherein said enzyme is an oxidoreductase.